Molecular mechanisms of bacterial translation regulation Open Access

Nguyen, Ha An (Fall 2021)

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Rapid and accurate translation to produce properly folded and functional proteins is essential to cell growth and survival. The ribosome is a complex macromolecular machine that directs the conversion of a nucleotide-based code (i.e. genes encoded in messenger RNA (mRNA)) into amino acids, the code of proteins. To accomplish this task, transfer RNAs (tRNAs) must accurately “decode” the genetic information on mRNA by pairing their anticodon sequences with mRNA codons, while carrying with them the specific corresponding amino acid. While the correct base pairing between the tRNA anticodon and mRNA codon is the fundamental driving force behind the fidelity of protein synthesis, it is also known that other elements of the tRNA as well as the ribosome also act as regulatory elements during protein synthesis. These additional layers of regulation are needed because the ribosome needs to quickly select the correct tRNA from the large pool of chemically and structurally similar tRNA molecules.

This dissertation analyzes how the ribosome maintains the three nucleotide mRNA frame and decoding fidelity. First, I investigated how the m1G37 modification and insertions of either G37.5 or A37.5 in tRNAPro sequence can circumvent recognition and proofreading by the ribosome enabling misreading and +1 frameshifting. After that, I studied structures of mismatched tRNA-mRNA pairs in both the ribosomal A and the P sites to uncover mechanisms with which the ribosome can recognize correct from incorrect tRNAs. Using tRNAAla bound to either a cognate or a near-cognate codon, I found that disrupting the evolutionarily correlated 32-38 pairing in the tRNA renders the ribosomal rRNA nucleotide A1913 unable to recognize the correct tRNA. Finally, I discovered an A-site mRNA positioning mechanism with which mismatches in the P site can trigger post-peptidyl transfer proofreading. Altogether, the biochemical and structural data presented here elucidate previously unappreciated mechanisms with which the integrity of the tRNA-mRNA pairing is regulated to ensure the accuracy and efficiency of gene expression.

Table of Contents

Abstract v

Acknowledgments vii

Table of Contents x

List of Figures xii

List of Tables xv

List of Abbreviations xvi

Chapter 1. 1

Introduction 1

1.1. Overview of translation 1

1.2. The ribosome undergoes large conformational changes during elongation 2

1.3. tRNA selection on the ribosome 5

1.4. tRNA elements contribute to accurate tRNA selection 7

1.5. Modifications and insertions in tRNAPro affect decoding and frame maintenance 11

1.6. The 32-38 base pair in tRNAs is an important recognition element 13

1.7. Post peptidyl transfer quality control 15

1.8. Figures 17

1.9. References 27

Chapter 2. 36

Importance of tRNA anticodon loop modification and a conserved, noncanonical anticodon stem pairing in for decoding 37

2.1. Abstract 38

2.2. Introduction 39

2.3. Results 43

2.4. Discussion 50

2.5. Experimental procedures 55

2.6. Figures and tables 59

2.7. Acknowledgments 73

2.8. References 73

Chapter 3. 80

Disruption of evolutionarily correlated tRNA elements impairs accurate decoding 80

3.1. Abstract 81

3.2. Significance 81

3.3. Introduction 82

3.4. Results 85

3.5. Discussion 89

3.6. Methods 92

3.7. Figures and tables 94

3.8. Acknowledgments 111

3.9. References 111

Chapter 4. 115

4.1. Abstract 116

4.2. Introduction 117

4.3. Results 121

4.4. Discussion 123

4.5. Materials and Methods 127

4.6. Figures and Tables 129

4.7. Acknowledgments 139

4.8. References 139

Chapter 5. 143

Conclusions and Future Directions 143

Figures 153

References 155

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